6-hydroxydopamine induces serotonergic axon sprouting in cerebral cortex of newborn rat

Newborn rats were administered the neurotoxin 6-hydroxydopamine (6-OHDA) to determine whether neonatal ablation of the noradrenergic (NE) innervation produces augmented growth (i.e., sprouting) of serotonergic (5-HT) raphe-cortical axons. Following NE denervation at birth, the density of 5-HT axons in motor cortex (AGI) was determined at 4 days postnatal. Using a computer microscope system, the positions of all 5-HT-positive axons were mapped in radial strips of cortex from treated and control rats. Cumulative axon length, expressed as a function of area inspected, was used as a parameter of innervation density. Following 6-hydroxydopamine, the cumulative length of 5-HT axons in motor cortex increases by 32% (P > 0.05) while cortical serotonin levels measured by HPLC concomitantly increase by 29% (P > 0.005). The combined increases in 5-HT axon density and in neurotransmitter levels indicate that NE denervation produces increased growth of the cortical 5-HT innervation by the 4th postnatal day. The amount of transmitter stored per unit length of 5-HT axons appears unchanged. In 6-OHDA-treated rats, 5-HT axons exhibit augmented growth in all layers of motor cortex. In the treated rats, the relative density of 5-HT axons in each cortical layer is roughly proportional to the normal innervation density. Accordingly, in motor cortex, the magnitude of 5-HT axon sprouting is greatest in layer VI, which normally receives a dense 5-HT innervation, and is less in layer V, which has a lower innervation density. Qualitative assessment of other cortical areas following 6-OHDA reveals that 5-HT axon density appears increased in cortical zones that normally receive a dense 5-HT innervation, while the density remains low in zones with sparse innervation. The absence of axonal sprouting is particularly striking in those zones which receive a dense NE innervation but are sparsely innervated by 5-HT axons. Thus, while 5-HT axons undergo sprouting, they do not appear to replace ablated NE terminals in areas with a sparse 5-HT innervation. Hence, normal laminar and regional specificity of 5-HT axons is preserved despite ablation of NE afferents. These data indicate that, while NE denervation may trigger serotonergic sprouting, competition between NE and 5-HT fibers for the same postsynaptic sites is not the main factor that regulates postnatal growth of these axonal projections. The present findings demonstrate that the early development of raphe-cortical projections is influenced by NE cortical innervation. NE axons appear to regulate the magnitude of 5-HT axon growth within the cerebral cortex without affecting regional selectivity of 5-HT axons for normal target sites. The demonstration of augmented 5-HT cortical innervation after NE depletion suggests that NE may normally inhibit growth of 5-HT axons in neonatal cortex.